1. Field of the Invention
The present invention relates to a coryneform bacterium that is able to efficiently produce a heterologous protein by secretory production and a method for secretory production of a heterologous protein. Specifically, the heterologous protein produced by secretory production according to the present invention is a multimeric protein.
2. Brief Description of the Related Art
To date, secretory production of heterologous proteins by microorganisms has been reported in Bacillus bacterium (Microbiol. Rev., 57, 109-137 (1993)), methanol-assimilating yeast, Pichia pastoris (Biotechnol., 11, 905-910 (1993)), filamentous fungi of the genus Aspergillus (Biotechnol., 6, 1419-1422 (1988) and Biotechnol., 9, 976-981 (1991)), and so forth.
Secretory production of heterologous proteins by coryneform bacteria has also been reported, specifically secretion of a nuclease and a lipase by Corynebacterium glutamicum (henceforth also abbreviated as C. glutamicum) (U.S. Pat. No. 4,965,197, J. Bacteriol., 174, 1854-1861 (1992)), secretion of a protease such as subtilisin (Appl. Environ. Microbiol., 61, 1610-1613 (1995)), secretion of a protein using signal peptides of cell surface layer proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria (Japanese Patent Laid-open (Kohyo) No. 6-502548), secretion of a fibronectin-binding protein using the signal peptide of PS2 (CspB) (Appl. Environ. Microbiol., 63, 4392-4400 (1997)), secretion of protransglutaminase using signal peptides of cell surface layer proteins PS2 (CspB) and SlpA (also referred to as CspA) of coryneform bacteria (Japanese Patent No. 4320769), secretion of a protein using a variant type secretion system (Japanese Patent Laid-open (Kokai) No. 11-169182), secretion of a protransglutaminase by a variant strain (Japanese Patent No. 4362651), secretion of a protein using a Tat-dependent signal peptide (Japanese Patent No. 4730302), and so forth.
Various proteins have been suggested as proteins that could be produced by secretory production; however, in coryneform bacteria, there are no reports of secretory production of any multimeric protein such as, for example, antibody-related molecules.
Metallopeptidases are a class of protease that requires various metal ions, such as zinc and calcium, for activation, and have an activity of decomposing various kinds of proteins. Among the metallopeptidases, metallopeptidases belonging to the M23/M37 family (also referred to as M23/M37 metallopeptidases) require zinc ion. It has already been elucidated that the Cgl0858 gene of C. glutamicum is a gene coding for a protein that includes a region homologous to a motif of M23/M37 metallopeptidases on the basis of the sequence information. However, the function of the protein encoded by the Cgl0858 gene in C. glutamicum is still elusive. Moreover, the ability to improve the production amount of an objective heterologous protein due to high expression of a gene coding for a region homologous to this motif of the M23/M37 metallopeptidases has not been reported for any biological species.
Penicillin-binding protein (PBP) is a generic term which describes proteins that bind with β-lactam type antibiotics, and as a result, inhibit binding with β-lactam type antibiotics. PBPs are generally membrane-binding proteins, and they are considered essential for cell wall synthesis of eubacteria. PBPs are classified as high molecular weight PBPs (HMW-PBPs) or low molecular weight PBPs (LMW-PBPs), according to the molecular weights thereof. HMW-PBPs are further classified as class A high molecular weight PBPs (class A HMW-PBPs), which have both a transpeptidase activity domain for crosslinking peptidoglycan moieties, and a transglycosylase activity domain for forming a polysaccharide chain from disaccharides, and class B high molecular weight PBPs (class B HMW-PBPs) which have only a transpeptidase activity domain.
The findings about PBPs of C. glutamicum are detailed in Mol. Microbiol., 66, 643-57 (2007), Antonie Van Leeuwenhoek, 94, 99-109 (2008), and so forth. In C. glutamicum, at least nine PBP homologues have been found so far. Five of them are HMW-PBPs including two class A HMW-PBPs (PBP1a, PBP1b) and three class B HMW-PBPs (FtsI, PBP2a, PBP2b). It is known that the class A HMW-PBPs of C. glutamicum are responsible for cell extension, and the class B HMW-PBPs are responsible for formation of peptidoglycan of septal walls at the time of cell division.
However, the relationship between penicillin-binding protein and the secretory production of a heterologous protein has not been previously reported.